GRASP (GFP Reconstitution Across Synaptic Partners), originally developed in invertebrates, is a genetically controllable fluorescence-based system for identifying sites of contact between two cells (or cell populations). It relies on the expression of membrane-tethered split (i.e. non-functional) GFP modules that must come together to reconstitute GFP fluorescence (spGFP1-10 &spGFP11);since this functional complementation requires close apposition between membranes (<100 nm), GRASP is a powerful system to identify synapses between two cells. The objective of this application is to develop and validate GRASP methods for use in mammalian and Drosophila neural circuits. We will generate a battery of GRASP tools, including viral vectors for targeted expression of spGFPs, produce transgenic (and knock-in) lines expressing spGFPs under the control of the Cre or tetracycline inducible systems, and engineer Drosophila chromosomes harboring GRASP components under orthogonal expression control systems. Together, these reagents will afford temporal and spatial selectivity of GRASP expression, and provide a versatile platform for circuit mapping. We propose to test these components in two different models: a thermosensory circuit screen in flies and a Cre reporter screen in mice. We will also engineer a multicolor GRASP as a novel "synaptic fingerprinting" technique. We expect the results of these studies to significantly enhance the arsenal of tools available for circuit mapping, and be helpful in the characterization of normal and diseased nervous systems. PUBLIC HEALTH RELEVANCE: Mapping the connectivity of neural circuits is an important prerequisite to understand how neuronal ensembles process sensory stimuli and drive behavior. Towards this goal, we will develop and validate new strategies to more easily identify synapses between defined neuronal populations in the mouse. We anticipate that inducible and multi-color GRASP, as well as the novel Grainbow transgenic lines and viral vectors will provide a toolbox that will be of considerable value for the entire neuroscience community.